PROJECT SUMMARY During mammalian preimplantation development, one of the most dramatic epigenetic events is the global erasure of DNA methylation (5-methylcytosine or 5mC) from the parental genomes. This reprogramming is critical to reset the methylation status of the gametes and restore developmental potency to pluripotent cells in the blastocyst. Elucidating the molecular mechanisms that regulate 5mC reprogramming is central to gaining deeper insights into normal embryonic development as well as to better understand aberrant 5mC patterns and imprinting disorders associated with assisted reproductive technologies (ART). While early studies showed that the paternal genome undergoes ?active? demethylation through conversion of 5mC to 5-hydroxymethylcytosine (5hmC) and the maternal genome undergoes ?passive? demethylation through a lack of maintenance DNA methyltransferase (Dnmt1) activity during replication, more recent studies have suggested that 5mC erasure from the parental genomes is a combination of these two demethylation pathways. Unraveling the mechanisms that regulate 5mC erasure has been challenging due to our inability to directly distinguish between these pathways. This limitation can be overcome by making genome-wide strand-specific measurements of 5mC in single cells. In addition, this epigenetic reprogramming coincides temporally with the first cell fate specification in the embryo towards the trophectoderm (TE) and inner cell mass (ICM) lineages. However, it remains unclear how 5mC reprogramming influences this cell fate decision. To address these questions, in Specific Aims 1 we propose to develop a novel single-cell sequencing technology to simultaneously quantify 5mC strand-specifically together with mRNA from the same cell. Preliminary experiments in mouse embryonic stem cells and mouse embryos show that we can detect both 5mC and mRNA from the same cell. To test our central hypothesis that the mechanisms regulating the erasure of 5mC are parent- and stage-specific, in Specific Aims 2 we plan to quantify the genome-wide strand-specific distribution of 5mC in hybrid mouse embryos from the 2- to 64-cell stage of embryogenesis. Further, through stochastic mathematical modeling and knockdown experiments, we will elucidate how different molecular factors regulate active and passive demethylation pathways during preimplantation development. Finally, while cell-to-cell variability in a variety of factors have been shown to bias the cell fate potential of early blastomeres towards the TE or ICM lineages, it remains unclear how 5mC reprogramming tunes or reinforces these decisions. To address this question, in Specific Aims 3 we plan to quantify both 5mC and mRNA from the same cell to directly correlate how heterogeneity in genome-wide strand- specific patterns of 5mC influence the decision between symmetric vs. asymmetric cell divisions and the resulting cell fate choices. Thus, through the development of novel single-cell methods we will gain a deeper mechanistic understanding of global demethylation dynamics during preimplantation development that will enable us to better study altered epigenetic reprogramming associated with ART and other environmental stresses in future.